The long-term goal of this project is to elucidate mechanisms underlying assembly of the enterocyte brush border: the sole site of nutrient absorption and the primary surface of interaction with bacteria and bacterial products that accumulate in the intestinal lumen. Found at the apex of the enterocyte, the brush border contains up to one thousand tightly packed, actin bundle-supported membrane protrusions known as microvilli, which extend off the cell surface to a nearly identical length. The functional consequence of this arrangement is an immense capacity for housing membrane-associated nutrient processing and host defense machinery that is required for maintaining gut homeostasis. Despite being positioned at a critical physiological interface in the GI tract, there s little information on how microvillar actin bundles are nucleated, how microvillar length is controlled, or how microvilli achieve perfectly tight packing during enterocyte differentiation. Using the CACO-2BBE cell culture model to explore the physical remodeling of the enterocyte apical surface during differentiation, our laboratory made a series of exciting discoveries that provide insight on fundamental mechanisms of brush border formation. During the early stages of brush border assembly, we observe that microvilli cluster together and interact at their tips to form 'tepee' shaped structures. As differentiation proceeds, the observed 'tepees' grow larger by incorporating more microvilli. Electron microscopy of these structures revealed, for the first time that adjacent microvilli in these tepees are physically connected to each other by thread-like links. These observations suggest that the tight packing of microvilli during brush border assembly may be driven by adhesion complexes that are inherent to these protrusions. We also identified a member of the cadherin superfamily, protocadherin-24 (PCDH24), which could play a role in forming inter-microvillar adhesion links. PCDH24 is a novel microvillar component that exhibits striking enrichment at microvillar tips (observed with super-resolution light microscopy and immuno- EM) and shRNA-mediated knockdown of this molecule significantly impairs microvillar packing during CACO- 2BBE differentiation. Based on these and other preliminary findings, we propose that PCDH24 creates inter- microvillar adhesion links at microvillar tips, which are required for the tight packing of these protrusions during brush border assembly. The Aims proposed herein will begin to test this hypothesis by investigating: (1) the targeting and requirement for PCDH24 during brush border assembly and effacement by enteric pathogens, (2) the adhesion capacity of PCDH24, and (3) the mechanism underlying the microvillar tip localization of PCDH24. Given our expertise in defining the biological and physical underpinnings of brush border function, our group is well positioned to test this hypothesis and generate novel insight on this fundamental aspect of GI epithelial biology.